The present invention is in the field of secreted proteins that are related to the microtubule-associated protein secreted subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Secreted Proteins
Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins.
Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a xe2x80x98signal sequence trapxe2x80x99 technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer.
Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991 March-November;256(2-6):127-38).
The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al., Ann N Y Acad Sci Sep. 24, 1993 695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer""s disease (Mattson et al., Perspect Dev Neurobiol 1998; 5(4):337-52).
Breast cancer cells secrete a 52K estrogen-regulated protein (see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis.
Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan, Adv Exp Med Biol 1978;102:105-19).
Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996 September;1(3):225-32).
Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secreted IgE proteins.
For a further review of secreted proteins, see Nilsen-Hamilton et al., Cell Biol Int Rep 1982 September;6(9):815-36.
Microtubule-Associated Protein 1A/1B Light Chain 3
Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. Microtubule-associated proteins 1A (MAP1A) and MAP1B are abundant neuronal MAPs thought to be involved in neurite formation and stabilization. MAP1A and MAP1B are two microtubule-associated proteins expressed at high levels in developing and mature neurons. These MAPs are synthesized as polyproteins that are proteolytically processed to form a heavy chain (xcx9c250 kDa) and a light chain (xcx9c30 kDa). Light chain 3 (xe2x80x9cLC3xe2x80x9d) is a subunit of both MAP1A and MAP1B.
Experimental data indicates that MAP1A stabilizes microtubules in postnatal axons. MAP1B is expressed in neurons and glial cells predominantly during a period of extension of neurites in the developing brain, suggesting an important role for MAP1B during neurite formation and axon guidance. The relative levels of MAP1A and MAP1B change dramatically during development, with MAP1B expression highest in forming neurons, and MAP1A expression highest in mature neurons.
Light chain 3 (LC3) is a subunit of the neuronal microtubule-associated proteins (MAPs), MAP1A and MAP1B. Recent findings show that the cDNAs for MAP1A and MAP1B encode polyproteins that contain the MAP1A or MAP1B heavy chain and an LC2 or LC1 subunit, respectively. The cDNA encoding rat LC3 has been sequenced, and its sequence is not found in the MAP1A/LC2 or MAP1B/LC1polyprotein cDNAs. The deduced amino acid sequence of LC3 is highly conserved between rat and mouse. Rat LC3 is a 16.4-kDa protein with a predicted pI of 9.2. It is encoded on a 1.7-kilobase mRNA. Purified recombinant rat LC3 retains the ability to associate with microtubules assembled in the presence of brain MAPs and with microtubules assembled from purified tubulin.
Anti-LC3 immunohistochemistry reveals that LC3 in rat brain is restricted to neurons that are expressing either the MAP1A or MAP1B heavy chain. Although LC3 is expressed exclusively in cells expressing heavy chains, developmental changes in the total amount of LC3 protein are not proportional to changes in the amount of either the MAP1A or MAP1B heavy chain. LC3 protein expression measured by quantitiative immunoblotting is twice as high in postnatal brain as in embryonic and adult brain. The localization of the LC3 gene to human chromosome 20cen-q13 demonstrates that LC3 is the only MAP1 subunit that is not linked to the heavy chain genes. Because LC3 is a component of both the MAP1A and MAP1B microtubule-binding domains, the heavy-chain independent regulation of LC3 expression might modify MAP1 microtubule-binding activity during development.
For more information on MAP1A/1B light chain 3, see: Mann et al., J Neurosci Res Mar. 1, 1996 43(5):535-44; and Mann et al., J Biol Chem Apr. 15, 1994 269(15):11492-7.
Secreted proteins, particularly members of the microtubule-associated protein secreted protein subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins. The present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the microtubule-associated protein secreted protein subfamily.
The present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the microtubule-associated protein secreted protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein.